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ELISA
Introduction Enzyme-linked immunosorbent assays (ELISA) are plate-based assays designed for detecting and quantifying biomolecules such as peptides, proteins, antibodies and hormones. In an ELISA, antigens or antibodies are bound to a solid surface (plate) and then complexed with an antibody (antibody-antibody specific interaction) that is linked to an enzyme. A substrate is added and converted by enzyme to a measurable colored product, the rate of color formation if proportional to the amount of specific antigen.The extreme specificity or discrimination power of antibodies to recognize infinite arrays of antigenic structures makes the application of ELISA to analytical extremely valuable.ELISA are generally performed as a solid-phase technique (96-well polystyrene plates), which will passively bind antibodies and proteins. ELISA technique can be classified into two main types; competitive and noncompetitive assays. The competitive assay uses either an antigen-enzyme conjugate or an antibody-enzyme conjugate. The noncompetitive assays uses a double antibody “sandwich” technique where the second antibody has an indicator enzyme conjugated to it In ELISA technique antigen or antibody (immunoreactants) is immobilized onto a microtiter plate surface by adsorption, via a noncovalent interactions, which makes it easy to separate bound from unbound antigen during period of washing after incubation. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antibody is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen. After further incubation and washing, the amount of antigen present is visualized by the addition of chromogenic or fluorogenic substrate, depending of the sensitivity and amplification system used (spectrophotometer, fluorometer or luminometer) ( Walker,1994, p.461). The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Origin and Development Prior to the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique that employ radioactively labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal, which indicates whether a specific antigen or antibody is present in the sample. In 1960 Rosalyn Sussman Yalow and Solomon Berson published first described radioimmunoassayRadioimmunoassay technique requires the use of radioactively labeled antigens or antibody, as such posses a potential health risk, thus requires a much safer and efficient alternative approach. In 1971 two research groups published the format of the ELISA independently and simultaneously; Peter Perlmann and Eva Engvall at Stockholm University, and Anton Schurs and Bauke van Weemen in the Netherlands. The assay was based on similar principles of conventional radioimmunoassay; the key difference is that the antibodies are labeled with enzyme, rather than radioisotopes. Enzyme-based ELISA test uses nanoparticles as a chromogenic reporter to produce color signal from the detection of attograms of analyte; blue colors indicate positive results and red color for negative Method Indirect ELISA (Sandwich ELISA) and direct ELISA : The words “direct” and“indirect” are commonly used to describe various ELISA methodologies, and they refer to differences in how the antigen is captured, or immobilized to wells of a microplate, and also how the captured antigen is then detected. With direct capture, the components of the sample are adsorbed nonspecifically to the plastic well surface of a 96 well polystyrene plate. With indirect capture, the wells are first coated with specific antibody that then selectively binds and captures only the specific antigen from the test sample. Whichever capture method is used, the antigen can then be detected either directly or indirectly. For direct detection, a primary antibody that is conjugated to a reporter such as horseradish peroxidase, or HRP, is incubated with the captured antigen. Once unbound antibody is washed off, an enzyme substrate is added to detect the antibody-antigen interaction. For indirect detection, an unlabeled primary antibody is incubated with the bound antigen. Then a secondary antibody conjugated to the reporter recognizes the primary antibody. Because multiple secondary antibodies bind to each primary antibody, this approach allows multiple reporter molecules to localize to each antigen, thereby amplifying the signal and increasing the sensitivity of antigen detection. The term “sandwich” ELISA is commonly used to refer to assays involving indirect capture, since the antigen is sandwiched between two specific antibodies. Most commercial ELISA kits are sandwich ELISAs that use indirect capture, indirect detection and avidin-biotin chemistry, in which avidin strongly binds to multiple biotin molecules. For ELISA, a biotinylated primary antibody is bound to the antigen, followed by avidin, streptavidin or other biotin-binding molecule that is conjugated to the reporter. Thus, multiple reporters are localized to the antigen resulting in signal amplification. Competitive ELISA: Unlike the Sandwich ELISA, which requires matched antibody pairs that recognize separate epitopes on the target antigen. The competitive ELISA detects target molecules using only one epitope. As the name indicates, this type of ELISA uses a labeled antigen to compete with the target antigen (sample antigen) in the sample for immobilization and detection. Many commercial competitive ELISA kits come with a 96-well plate that is pre-coated with a secondary antibody. The sample containing the target antigen is combined with purified antigen that has been labeled with a reporter, such as alkaline phosphatase, and a primary antibody that recognizes the target antigen, and the mixture is incubated in the plate. The unlabeled and labeled antigens compete for binding to the primary antibody; the primary antibody in turn binds to the immobilized secondary antibody. After washing the plate, the amount of labeled antigen bound to the plate is then detected. Because competitive binding is concentration dependent, the signal output is inversely correlated with the amount of antigen in the sample. Therefore, high antigen concentration, implies low signal output; low antigen concentration, implies high signal output. Types of ELISA Noncompetitive or Sandwich Assay Antibody measuring system o Titer wells coated with suitable antigen o Add target sample (patient blood) containing the antibody o Incubate, till antigen antibody reaction is complete o Wash off unbound antibody o Add antibody labeled with enzyme o Incubate till labeled antibody binds antigen-antibody complex o Wash to remove unbound labeled antibody o Add colorless substrate enzyme, then measure color o Color proportional to antibody in target sample Competitive binding assay o Titer-wells coated with antibodies o Known quantities of patient blood or target sample containing antigen + antigen labeled with enzyme o Incubate, till antigen antibody reaction is complete o Wash to remove unbound antigens o Add substrate, then incubate o Enzyme + substrate ->product -> measure color o Color inversely related to antigen in target sample or patient blood sample. Uses/Application of ELISA Enzyme-linked immunosorbent assay, is a biochemical technique used mainly in immunology to detect the presence of antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. ELISA has many application, it can be performed to evaluate either the presence of antigen or the presence of antibody in a sample; it is a useful tool both for determining serum antibody concentration, and also for detecting the presence of antigen. It also has applications in the food industry in detecting potential food allergens such as milk, almonds, eggs, and peanuts.ELISA uses include: (1) measure antibody levels e.g. vaccines (2) detect viruses e.g. HIV, Hepatitis, etc. (3) detect hormonal changes, like in the case of pregnancy (4) detect circulatory inflammatory markers, e.g. cytokines References Hayworth, D. (2014, January 1). Overview of ELISA. Retrieved October 1, 2014 Van Weemen BK & Schuurs AHWM (1971) Immunoassay using antigen—enzyme conjugates. FEBS Letters 15(3):232-236 Walker, J. (1994). P.461. In Basic protein and peptide protocols. Totowa, N.J.: Humana press Wide L, Porath J. Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies. Biochem Biophys Acta 1966; 30:257-260